How xMAP® Technology Works

Luminex® xMAP® technology enables scientists to measure multiple proteins in a single well. This technology combines advanced fluidics, optics, and digital signal processing with proprietary microsphere technology to deliver multiplexed assay capabilities. Featuring a flexible open-architecture design, xMAP® technology can be configured to perform a wide variety of bioassays quickly, cost-effectively and accurately.

Microsphere beads, either polystyrene or paramagnetic, are color-coded into up to 500 distinct sets. Each bead set can be coated with a reagent specific to a particular bioassay, allowing the capture and detection of specific analytes from a sample. Inside the Luminex® analyzer, a light source excites the internal dyes that identify each microsphere particle, and also any reporter dye captured during the assay. Multiple readings are made on each bead set, which further validates the results. Using this process, xMAP® technology allows multiplexing of up to 500 unique bioassays within a single sample, both rapidly and precisely. xMAP® technology is compatible with the following Luminex® analyzers:

  • MAGPIX® system—affordable, efficient, and compact
  • Luminex® 100/200™ system—versatile, efficient, and widely used in multiplexing
  • FLEXMAP 3D® system—high throughput, up to 500 simultaneous assays, and automation compatible

Novex® multiplex immunoassay tips and tricks

Multiplex Kits

Our multiplex kits undergo rigorous quality testing (Table 1) and are calibrated against matching Novex® ELISA
kits, if available, so that both protein analysis platforms will provide comparable analytical results. Novex®
multiplex kits are:

  • Fast and efficient—simultaneous analysis of multiple proteins using only 50 μL of precious sample
  • Accessible—broad and expanding menu of Novex® immunoassay kits based on xMAP® technology
  • Economical—can significantly reduce time and costs compared to running multiple western blots or ELISA assays

Life Technologies makes the use of Luminex® xMAP® technology simple and reliable by providing a range of Novex® singleplex and multiplex immunoassay kits, a full line of multiplexing Luminex® instruments, xPONENT® data analysis software, and extensive assay and instrument technical support, including on-site demonstrations and personalized technical consultation services.

Specification Description
Benchmarking to ELISA (see Figure 1)Correlates to ELISA data (>90% correlation)
RecoveryTested on serum and plasma
SensitivityPhysiologically relevant levels, <10 pg/mL (based on detectable signal >2 SD above background)
Precision (see Figure 2)
  • Inter-assay CV: <10%
  • Intra-assay CV: <10%
Specificity (see Table 2)Cross-reactivity tests are performed with other analytes and antibodies
Linearity of dilutionHigh coefficient of correlation between sample dilutions and expected concentration over the range
of the assay
Parallelism to natural samples (see
Figure 3)
(R2 > 0.99) Recombinant standards are compared to natural samples to ensure equivalency

 

Table 1. Rigorous assay validation of Novex® multiplex kits helps ensure consistent, reliable results.

 

How to prepare your Novex® multiplex assay

 

Figure 1. Multiplex assay results correlate to ELISA assay data. Murine GM-CSF in tissue culture supernatant fluid was tested by ELISA and in a Novex® multiplex assay. Correlation of values over 3 logs of sample dilution was 0.9868.

 

Figure 2. Assay precision. Green and gray bars each represent 24 replicates measured on separate days. CVs in all cases were less than 10%. Data were generated using a Novex® human cytokine 10-plex magnetic assay kit.

How to analyze multiplexed protein data

 

Figure 3. Parallelism of recombinant standards to natural samples. To evaluate the Novex® multiplex assays, samples of 23 human protein markers were spiked into a sample of human serum, and the sample was processed using the manufacturer’s instructions for the Novex® multiplex assay kit and using a similar kit from two other suppliers. The multiplex sample was quantified on the MAGPIX® system, and percent recovery calculated (A) for the individual markers and (B) as an average for the entire group.

 

Antibody Bead Kit

 IL-1ϐIL-2IL-4IL-5IL-6IL-8IL-10TNF-αIFN-γGM-CSF
IL-1ϐ6,33241371393732271851
IL-23811,3244717264033271550
IL-4384110,50315254032251250
IL-54854368,265113433251651
IL-6447240116,8274231221270
IL-8393944122710,00232271952
IL-104151391112418,151231246
TNF-α32553610760306,6421047
IFN-γ3241391087035253,54051
GM-CSF414036128463325134,932

 

Table 2. Specificity data from the human cytokine 10-plex panel shows no measurable cross-reactivity. Numbers represent mean fluorescence intensity (MFI) units generated when individual recombinant proteins were analyzed independently in a series of 10-plex assays.